The first specific aim of the previously funded research proposal was to characterize secretory granules within living primary-cultured, pancreatic beta cells at rest and upon stimulation. A primary reason for this renewal application is the considerable effort and time required to establish a reliable method for preparing primary-cultured beta cells. With that method established, an additional year of support should be sufficient time to complete the work outlined below. The work included in this request for continued funding will expand upon a very intriguing observation made during the first period of support. That is, upon stimulation with both potassium and glucose, fluorescent-labeled secretory vesicles in primary-cultured pancreatic beta cells brighten substantially while apparently remaining inside of the cell. This observation is quite unlike the response seen in chromaffin cells where stimulation leads to the rapid disappearance of the fluorescently labeled vesicles. These results suggest a fundamental difference in the secretion mechanisms of two closely related cell types. Additionally, in response to glucose steps, the pattern of these vesicle brightening appears pulsatile. With TIRF microscopy, it is possible to quantify pulse mass and frequency at the single granule level. The results of the studies outlined in this proposal will provide key insight into insulin secretion at the single cell level.